Advanced Multifunctional Hydrogels for Enhanced Wound Healing through Ultra‐Fast Selenol‐SNAr Chemistry

Abstract Fabrication of versatile hydrogels in a facile and effective manner represents a pivotal challenge in the field of biomaterials. Herein, a novel strategy is presented for preparing on‐demand degradable hydrogels with multilevel responsiveness. By employing selenol‐dichlorotetrazine nucleophilic aromatic substitution (SNAr) to synthesize hydrogels under mild conditions in a buffer solution, the necessity of additives or posttreatments can be obviated. The nucleophilic and redox reactions between selenol and tetrazine culminate in the formation of three degradable chemical bonds—diselenide, aryl selenide, and dearomatized selenide—in a single, expeditious step. The resultant hydrogel manifests exceptional adaptability to intricate environments in conjunction with self‐healing and on‐demand degradation properties. Furthermore, the resulting material demonstrated light‐triggered antibacterial activity. Animal studies further underscore the potential of integrating metformin into Se‐Tz hydrogels under green light irradiation, as it effectively stimulates angiogenesis and collagen deposition, thereby fostering efficient wound healing. In comparison to previously documented hydrogels, Se‐Tz hydrogels exhibit controlled degradation and drug release, outstanding antibacterial activity, mechanical robustness, and bioactivity, all without the need for costly and intricate preparation procedures. These findings underscore Se‐Tz hydrogels as a safe and effective therapeutic option for diabetic wound dressings.


Model reaction of selenol and dichloromethyltetrazine
N-propylamine (118 mg, 2 mmol) was dissolved in 10 mL THF, oxygen was removed by argon for 10 min, and then γ-selenbutylactone (315 mg, 2.1 mmol) was injected into the syringe, stirring at room temperature for 8 h.Dichlorometetrazine (166 mg, 1.1 mmol) was added to the above solution, stirring reaction was carried out, and TLC was followed up.After spinning the solvent and dissolving it with deuterated chloroform (CDCl3) as solvent, nuclear magnetic hydrogen spectrometry and high performance liquid chromatography were performed.

Synthesis of SeH and PEG-Se2 hydrogels
The preparation of 4-arm polyethylene glycol with selenol (4-arm-PEG-SeH) followed the protocol outlined in our earlier research.One gram of 4-arm-PEG-NH2 was added to 4 mL of deionized water.Thereafter, SBL was added to the solution at a molar ratio of 4:1 (SBL to 4-arm-PEG-NH2), followed by continuous stirring at 25°C for 8 h, resulting in the successful synthesis of 4-arm-PEG-SeH.The solution was placed in the air for oxidative crosslinking for 24 h to prepare PEG-Se2 hydrogels.

Rheological measurements
All rheological measurements were performed at 37 °C to simulate the human environment.The distance between the rotor and the sample stage was 1 mm when using a 25-mm parallel plate for measurement and testing.The strain sweep settings were as follows: frequency, 1 Hz; strain sweep,1-3000%.The frequency sweep settings were as follows: strain, 1%; frequency sweep, 1-100 Hz.The time sweep test was performed at a frequency of 1 Hz, strain of 1% and sweep time of 300 s.In addition, the self-healing performance of the hydrogels was tested via rheological analysis.The hydrogels were scanned for 120 s alternately under strain conditions of 1% and 3000%, with a total of 3 cycles.After a cycle was completed, another cycle of 120 s was run.For the low-shear measurement at 0.1% strain, the frequency remained constant at 10 Hz during the entire process.

Hydrogels' antibacterial characteristics in vitro
Hydrogels and Tz (Tz, Se-Tz and Se-Tz@Met groups containing 0.25 mmol mL -1 of cyano group) were introduced into 1 mL of bacterial solution (S. aureus, E. coli and MRSA, 1 × 10 7 CFU mL -1 ), and the mixture was either subjected to green light (520 nm, 1 W cm -2 ) or placed in darkness.Subsequently, the solution was diluted 1000 times, and 50 μL of the solution was extracted before being placed onto a culture plate.After 16 h of culture, images were captured and analyzed.

Biocompatibility assay
The biocompatibility of hydrogels and Tz was evaluated viacalcein acetoxymethyl ester (calcein-AM) and propidium iodide (PI) doublestaining and methylthiazoletetrazolium (MTT) assays.First, double labeling with calcein-AM and PI, as reported earlier, was performed to determine cell viability. 2NIH-3T3 and HUVEC cells were treated with hydrogels and Tz before incubation at 37 °C for 24 h (all Se-Tz and Tz contained 0.25 mmol•mL -1 cyano group).Samples in the groups treated with green light (GL) were irradiated with GL (520 nm, 1 W cm -2 , 15 min, VCLHLGD0025017, Blueprint, Beijing, China).NIH-3T3 and HUVEC cells were stained for 40 min with red (PI) and green (calcein-AM) fluorescent dyes at 37 °C and imaged under a live cell imaging system (DMI6000B; Feica, Wetzlar, Germany).Second, cell viability was evaluated via the MTT assay.Briefly, NIH-3T3 and HUVEC cells were seeded in a 96-well plate (5 × 10 3 cells/well).Following cell attachment, the hydrogels (0-781 μg•mL -1 ) and Tz (0-60 mg•mL -1 ) were added to the cells.The GL-treated groups were illuminated with GL (520 nm, 1 W cm -2 , 15 min, VCLHLGD0025017, Blueprint, Beijing, China).After 24 h of incubation, the cells were washed and incubated for 4 h at 37°C in fresh medium containing MTT reagent.Finally, the absorbance of formazan crystals dissolved in DMSO was measured at 490 nm using a microplate reader (SpectraMax M3, Molecular Devices, California, USA).

Cell proliferation and migration assays
NIH-3T3 purchased from ATCC were cultured in DMEM medium supplement with 10% FBS and 1% penicillin−streptomycin and kept in a 37 °C humidified incubator contained 5% CO2.The NIH-3T3 and HUVEC proliferation stimulated by Se-Tz@Met scaffold dressing was evaluated by MTT.For cell migration assay and tube formation assay, NIH-3T3 and HUVECs were firstly co-cultured with FEP@exo hydrogel in the upper chamber of the transwell insert and culture medium with 1% FBS was added into the lower chamber.After 24 h of culture, the cells on the membrane of the upper chamber were carefully removed with a cotton swab.Then, cells migrated into the lower chamber was fixed with 4% paraformaldehyde and stained with crystal violet.An inverted microscope (Nikon, FHEIPSE Ti, Japan) was used to observe the stained cells.

Figure S3 .
Figure S3.Degradation process of Se-Tz small molecule.(a) he photodegradation process of Se-Tz small molecule tracked by HPLC.(b) Degradation kinetics of Se-Tz.(c) 13 C NMR spectrum of photodegradation products.(d) FT-IR spectra of photodegradation products and Se-Tz (e) GCT-TOF-MS of photodegradation products.

Figure S4 .
Figure S4.Properties of hydrogels obtained by reaction of selenol with DT in different proportions.(a) Nomenclature and state of reactions of different proportions of selenol with dichlorometetrazine.(b) XPS spectra of PEGSe2, P1, P2, P3 and P4.(c) The infrared spectrum of 4arm-PEG-NH2, 4arm-PEG-SeH and P1.(d) Rheological behaviors of hydrogels.Due to an excessive presence of monosubstituted tetrazine in the polymer structure at a 2:1 ratio, the gelation ability of the reaction system diminishes significantly, thus limiting further discussion on this matter within the scope of this paper